DESCRIPTION: These investigations will focus on elucidating the process of mRNA degradation in Escherichia coli. The goal of these studies will be to identify and characterize RNA elements responsible for differences in mRNA stability, to define the features of a key bacterial ribonuclease that determine its specificity and function, and to elucidate important aspects of the mechanism by which this ribonuclease acts. Molecular biological, biochemical, and genetic methods will be employed. Particular attention will be devoted to RNA degradation by RNase E, a ribonuclease thought to control the principal pathway for mRNA decay in E. coli. First, we will examine how the rate of internal RNA cleavage by this endonuclease is controlled by the phosphorylation state of the RNA 5' terminus. In addition, we will investigate the RNA and protein elements that govern the ability of RNase E to autoregulate its synthesis in E. coli, taking advantage of a powerful and convenient genetic system that we have developed for this purpose. Finally, we will examine the role of 5'-terminal RNA secondary structure as an impediment to mRNA degradation by the related E. coli endonuclease CafA. The results of these studies should enhance our knowledge of a fundamental aspect of gene regulation that presently is poorly understood. This knowledge should ultimately be of value in maximizing bacterial production of medically useful proteins and in clarifying a biological regulatory mechanism that can play an important role in microbial pathogenesis.